Host induced gene silencing targeting Aspergillus flavus aflM reduced aflatoxin contamination in transgenic maize under field conditions

Authors: RARUANG, YENJIT - Louisana State University; OMOLEHIN, OLANIKE - Louisana State University; HU, DONGFANG - Louisana State University; Wei, Qijian - Mei Mei; HAN, ZHU-QIANG - Guangxi Academy Of Agricultural Sciences; Rajasekaran, Kanniah - Rajah; Cary, Jeffrey; WANG, KAN - Iowa State University; CHEN, ZHI-YUAN - Louisana State University

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/30/2020
Publication Date: 4/28/2020
Citation: Raruang, Y., Omolehin, O., Hu, D., Wei, Q., Han, Z.-Q., Rajasekaran, K., Cary, J.W., Wang, K., Chen, Z.-Y. 2020. Host induced gene silencing targeting Aspergillus flavus aflM reduced aflatoxin contamination in transgenic maize under field conditions. Frontiers in Microbiology. 11:754. https://doi.org/10.3389/fmicb.2020.00754.
DOI: https://doi.org/10.3389/fmicb.2020.00754

Interpretive Summary: Aflatoxin contamination of maize and other food supplies results in hundreds of millions of dollars in crop loss annually, and globally imposes a severe health risk to exposed populations. The consumption of tainted crops can lead to liver cancer, stunted growth in children, and eventually death. Currently no single method of remediation has proven fully successful, thus the development of pre-harvest technologies is vital in reducing the effects of this toxigenic fungus. Our research demonstrated it is possible to reduce aflatoxin accumulation and Aspergillus flavus growth in maize using RNAi technology. RNAi molecules are small fragments of RNA that are generated in plants, animals and other eukaryotes. Once produced, these molecules can then recognize and bind to specific genes that are also being produced by the invading fungus. The binding of this RNAi molecule results in degradation of the target gene. We have exploited this natural system by generating transgenic maize plants that produce small RNAi molecules designed to target one of the genes responsible for making the toxin, aflM, in the fungus, A. flavus and degrade it resulting in significant reduction of aflatoxin levels. Further, since this method that generates the small RNAi molecules does not generate a transgenic protein in crops thus reducing the public concerns regarding GMO. These findings are useful to corn breeders, biotechnologist, and corn food industry to reduce aflatoxin-related toxicity and illness in animals and humans.

Technical Abstract: Maize (Zea mays L.) is one of the major crops susceptible to Aspergillus flavus infection and subsequent contamination with aflatoxins, the most potent naturally produced carcinogenic secondary metabolites. This pathogen can pose serious health concerns and cause severe economic losses due to FDA regulations on permissible levels of aflatoxins in food and feed. Although biocontrol has yielded some successes in managing aflatoxin contamination, enhancing crop resistance is still the preferred choice of management for long-term sustainability. Hence, host induced gene silencing (HIGS) strategy was explored in this study. The A. flavus gene aflM encoding versicolorin dehydrogenase, a key enzyme involved in the aflatoxin biosynthetic pathway, was selected as a possible target for suppression through HIGS. An RNAi vector containing a portion of the aflM gene was constructed and introduced into immature B104 maize zygotic embryos through Agrobacterium transformation. PCR analysis of the genomic DNA from leaf tissue confirmed the presence of the transgene in six out of the seven events. The lines that showed reduced aflatoxin production in laboratory aflatoxin kernel screening assays have been increased from T1 to T4 generation in the past four years. Changes in aflatoxin resistance in these resulting homozygous transgenic kernels have been evaluated under both field and laboratory conditions. The T2 generation kernels containing the transgene from two events out of four examined had less aflatoxin (p=0.01 and p=0.08) than those without the transgene. Field-inoculated homozygous T3 and T4 transgenic kernels also revealed lower levels of aflatoxins (p=0.04) than kernels from the null (segregated non-transgenic samples) or B104 controls. A similar result was observed when the harvested T3 and T4 homozygous transgenic kernels were evaluated under kernel screening assay conditions without inoculation (p=0.003-0.05). Two events were crossed with LH195, LH197, LH210 and PHW79 elite breeding lines and the resulting crosses supported less aflatoxin (p=0.02) than the crosses made with non-transgenic lines. In addition, significantly higher levels of aflM gene-specific small RNAs were detected in the transgenic leaf and kernel tissues, indicating that HIGS target aflM reduces aflatoxin in maize the enhanced aflatoxin resistance in the homozygous transgenic kernels is likely due to suppression of aflM expression through HIGS.